One aspect of genetic engineering involves the insertion of foreign DNA sequences derived from eucaryotic sources into Escherichia coli or other microorganisms. A further refinement of genetic engineering concerns inducing the resulting microorganism to produce polypeptides encoded by the foreign DNA. Production of polypeptides can be considered a two-step process, with each step including numerous substeps. The two steps are transcription and translation. To produce a polypeptide efficiently and in quantity both steps of the process must be efficient. Transcription is the production of mRNA from the gene (DNA). Translation is the production of polypeptide from the mRNA.
A critical substep of the transcription process is initiation, that is, the binding of RNA polymerase to a promoter-operator region. The sequence of deoxyribonucleotide bases which make up the promoter region may vary and thereby effect the relative efficiency of the promoter. The efficiency depends on the affinity of the RNA polymerase for the promoter.
The efficiency of translation is affected by the stability of the mRNA. Increased stability of the mRNA permits improved translation. Although the exact determinants of mRNA stability are not precisely known, it is known that mRNA secondary structure as determined by the sequence of its bases has a role in stability.
The initial substep of translation involves binding of the ribosome to a base sequence on the mRNA known as the Shine-Dalgarno sequence or the ribosomal binding site (RBS). The synthesis of polypeptides begins when the ribosome migrates along the mRNA to the AUG start codon for translation. Generally these codons are found approximately 10 bases "downstream" from the Shine-Dalgarno site. Factors which increase the efficiency of translation include those which enhance binding of the ribosomes to the Shine-Dalgarno site. It has been shown that the structure of the mRNA in the region of the Shine-Dalgarno sequence and the AUG codon and the distance between the Shine-Dalgarno sequence and the AUG codon each play a critical role in determining the efficiency of translation. Other factors which affect the efficiency of translation are premature termination and attenuation. Efficiency of translation can be improved by removing the attenuation sites.
A difficulty encountered in attempts to produce high amounts of eucaryotic polypeptides in bacterial cells involves the inability of cells producing large amounts of mRNA to grow efficiently. This difficulty can be eliminated by preventing transcription by a process known as repression. In repression genes are switched off due to the action of a protein inhibitor (repressor protein) which prevents transcription by binding to the operator region. After microorganisms have grown to desired cell densities, the repressed genes are activated by destruction of the repressor or by addition of molecules known as inducers which overcome the effect of the repressor.
Numerous reports may be found in the literature concerning the cloning of eucaryotic genes in plasmids containing the P.sub.L promoter from .lambda. bacteriophage. (Bernard, H. V., et al., Gene (1979) 5, 59; Derom, C., et al., Gene (1982) 17, 45; Gheysen, D., et al., Gene (1982) 17, 55; Hedgpeth, J. et al., Mol. Gen. Genet. (1978) 163, 197; Remaut, E., et al., (1981) Gene 15, 81 and Derynck, R., et al., Nature (1980) 287, 193. In addition, European Patent Application No. 041,767, published Dec. 16, 1981, describes expression vectors containing the P.sub.L promoter from .lambda. bacteriophage. However, none of these references describe the use of the C.sub.II ribosomal binding site.
The use of a vector containing the P.sub.L promoter from .lambda. bacteriophage and the C.sub.II ribosomal binding site has been described. (Oppenheim, A. B. et al., J. Mol. Biol. (1982) 158, 327 and Shimatake, H. and Rosenberg, M., Nature (1981) 292, 128.) These publications describe the production of increased levels of C.sub.II protein but do not involve or describe the production of eucaryotic proteins.
Other vectors which contain the P.sub.L promoter and the C.sub.II ribosomal binding site have also been described (Courntey, M. et al., PNAS (1984) 81, 669-673; Lautenberger, J. A. et al., Gene (1983) 23, 75-84 and Lautenberger, J. A. et al., Science (1983) 221, 858-860). However, all of these vectors lead to the production of fused proteins which contain the amino terminal portion of the C.sub.II protein.
In 1982 Shatzman and Rosenberg presented a poster at the 14th Miami Winter Symposium (Shatzman, A. R. and Rosenberg, M., 14 Miami Winter Symposium, abstract p98 [1982]). This abstract provides a non-enabling disclosure of the use of a vector containing P.sub.L from .lambda. bacteriophage, Nut and the C.sub.II ribosomal binding site to synthesize a "eucaryotic" polypeptioe (SV40 small T antigen is actually not a eucaryotic polypeptide but a viral protein) in an amount greater than 5% of the cell protein in an unnamed bacterial host. The operator used is not defined. Neither an origin of replication nor a gene for a selectable phenotype is identified. This system with which the vector is used is described as including certain host lysogens into which the vector can be stably transformed.
Applicants are aware of the existence of a pending U.S. patent application in the name of M. Rosenberg filed under Ser. No. 457,352 by the National Institutes of Health, Dept. of Health and Human Services, U.S.A. Portions of this application have been obtained from the National Technical Information Service, U.S. Dept. of Commerce. However, the claims are not available and are maintained in confidence. The available portions of the application have been reviewed. This disclosure is not enabling. It indicates that the host is important (p8, line 17) but fails to identify any suitable host. It further depends upon the use of a .lambda. mutant which is not specified (p4, line 20). It indicates that the host contains lysogens (p8, line 18) unlike the present invention in which the host is not lysogenic. It mentions cloning and expression of a eucaryotic gene, monkey metallothionein gene, (p7, line 18) but does not provide details. It specifies that neither the sequence nor the position of any nucleotide in the C.sub.II ribosomal binding region has been altered (p3, line 27).
Pending, co-assigned U.S. patent application Ser. No. 514,188, filed July 15, 1983, describes novel vectors useful for the expression of polypeptides in bacteria. These vectors include P.sub.L O.sub.L, N utilization site for binding antiterminator N protein, ribosomal binding site, ATG codon, restriction enzyme site for inserting the gene encoding the desired polypeptide, an origin of replication and a selectable marker. In these vectors the distance between the N utilization site and the ribosomal binding site is greater than about 300 base pairs. In addition, each of these vectors contains a specific ribosomal binding site which cannot be readily replaced. These vectors were not equally useful for expression of different polypeptides.
T.sub.1 T.sub.2 rRNA transcription termination sequences have been described (Brosius, J., et al., J. Mol. Biol. 148, 107 (1981)). The placement of T.sub.1 T.sub.2 rRNA termination sequences at the 3' end of a procaryotic gene and the expression of such gene under the control of a promoter have been described (Amann, E., et al., Gene (1983) 25, 167; Zabeau, M., et al., The EMBO Journal (1982) 1, 1217).
European patent application no. 81304573.9, published April 14, 1982 under European publication no. 049,619, discloses the use of the .lambda.cI857 thermoinducible repressor as a stabilizing element. The repressor is cloned on the plasmid. A .lambda.cI90 prophage defective in repressor synthesis is introduced by infection. The prophage is maintained by the cloned repressor at temperatures below 32.degree. C. Any cell losing the plasmid will be lysed. If the temperature is increased to above 38.degree. C., the repressor is destroyed or inactivated and the cells lyse. This stabilization system is not compatible with the vectors of the invention which include .lambda.P.sub.L promoter and which express polypeptides at temperatures above 38.degree. C.
Origins of replication from constitutive high copy number plasmids are known. For example pOP1.DELTA.6 origin of replication from ColEl has been described (Gelfand, D. H., et al., PNAS (1978) 75, (12), 5869 and Muesing, M., et al. Cell (1981) 45, 235). In addition, high copy number run-away replication plasmids, as distinguished from, constitutive high copy number plasmids, are known (Remant, E., et al. Gene (1983) 22, 103).
The present invention relates to expression vectors which unexpectedly provide enhanced expression of different polypeptides. By employing different ribosomal binding sites in the vectors of this invention it is possible to achieve enhanced expression levels of different polypeptides relative to the levels achieved with the previous vectors. In addition, using the same ribosomal binding sites as in the previous vectors, it is possible to achieve enhanced expression of the same polypeptides. Moreover, by placing T.sub.1 T.sub.2 rRNA termination sequences at the 3' end of the gene encoding a polypeptide whose expression is desired, it is possible to increase the amount of desired polypeptide relative to the total polypeptide produced by a bacterial host. As importantly, the presence of the T.sub.1 T.sub.2 rRNA transcription termination sequences permit origins of replication derived from constitutive high copy number plasmids to be incorporated into expression vector without loss of the ability to replicate in constitutive high copy number.
Origin of replication derived from pBR322 or nonconstitutive high copy number plasmids other than runaway high copy number plasmids when incorporated into a vector are capable of producing only a certain number of copies per cell, typically less than 40 copies per cell. By substituting an origin of replication from a constitutive high copy number plasmid it has unexpectedly been found that that level of polypeptide expression is increased.
The preferred vectors of this invention are stabilized in the bacterial host and when bacteria containing plasmids which include the vectors and genes encoding polypeptides are grown, the plasmids are not lost. In this way, yield reduction caused by plasmid instability is overcome. Moreover, use of such preferred vectors avoids the use of antibiotic resistance as a selectable marker, thus permitting lower costs for producing polypeptides.